Amyloidogenic 60–71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects.

In this study, we investigated the impact of naturally occurring amyloidogenic mutations Δ60-71/ValThr insertion (Δ60-71/VT) 26 , Δ70-72 27 , and F71Y 28 occurring around the third aggregation-prone segment (residues 67-72) on the fibril-forming properties of the N-terminal 1-83 fragment of apoA-I.Our kinetic and thermodynamic analyses revealed that the Δ60-71/VT mutation generates a new aggregation-prone segment around residues 53-62, which promotes the nucleation process through reducing entropic barrier, thereby facilitating the aggregation and fibril formation of the apoA-I 1-83 fragment.

Effects of amyloidogenic mutations of structure and fibril-forming properties of full-length apoA-I
We initially examined the effects of the Δ60-71/VT, Δ70-72, and F71Y mutations on the structure and stability of full-length apoA-I.Circular dichroism (CD) spectra indicated that all apoA-I variants exhibited similar α-helical structures (Fig. 1A).However, thermal unfolding measurements, monitored by ellipticity at 222 nm, demonstrated a decrease in the thermal stability of apoA-I in the amyloidogenic variants, particularly in Δ60-71/ VT and Δ70-72 (Fig. 1B and Table 1).Consistently, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectra, which reflect the exposure of hydrophobic surfaces of proteins 29 , exhibited a significant increase in amyloidogenic variants in the following order of Δ70-72 > Δ60-71/VT > F71Y (Fig. 1C and Table 1).These findings indicate that the amyloidogenic variants, particularly Δ60-71/VT and Δ70-72, considerably destabilize the N-terminal α-helix bundle structure of apoA-I 3,12 .A thioflavin T (ThT) fluorescence assay demonstrated that none of the full-length amyloidogenic variants exhibited a propensity to form amyloid fibrils, similar to the N-terminal 1-83 fragment of apoA-I (Fig. 1D).This indicates that the destabilization of the protein structure by amyloidogenic mutations does not promote apoA-I fibril formation at neutral pH 12 .www.nature.com/scientificreports/reduces the lag time and increases the apparent rate constant for fibril elongation compared to the 1-83 fragment (Fig. 2D, E).Kinetic analysis according to the Finke-Watzky two-step model, in which homogeneous nucleation is followed by autocatalytic heterogeneous fibril elongation 30,31 provided similar conclusions: the Δ60-71/VT mutation significantly increases both the rate constants of nucleation and fibril elongation in fibril formation (Supplementary Fig. S1).Secondary structural changes in the 1-83 variants during incubation were assessed using CD measurements.As shown in Fig. 3, all 1-83 variants, which existed as random coil structures before incubation, exhibited single minimum spectra at approximately 216 nm after incubation for 120 h, implying conversion to a β-sheet-rich structure.Atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and transmission electron microscopy (TEM) revealed that all the 1-83 variants formed ThT reactive thin and straight fibrils after incubation (Fig. 4A).However, despite the similar apparent morphology of fibrils formed by the amyloidogenic variants, the fibrils of the Δ60-71/VT variant exhibited higher stability against urea-induced disaggregation than the other variants (Fig. 4B and Supplementary Fig. S2).This suggests the presence of differences in the β-sheet structure of the Δ60-71/VT variant.In addition, the fibrils formed by all amyloidogenic 1-83 variants and the wild-type fragment induced similar cytotoxicity in HEK293 cells (Supplementary Fig. S3), which is consistent with previous findings that the formation of fibril structures is essential for the cytotoxicity of apoA-I 1-83 variants 12,25 .

Effects of monomer concentration and seed fibrils on fibril-forming kinetics of apoA-I 1-83 variants
Next, we investigated the dependence of fibril-forming kinetics on the monomer concentration of the apoA-I 1-83 variants.Figure 5A, B display the time courses of ThT fluorescence intensities for the apoA-I 1-83 and Δ60-71/VT variants at various initial monomer concentrations.Previous reports have demonstrated that the half time of fibril formation depends on the initial monomer concentration, indicating that either nucleation or fibril elongation is the dominant step 32,33 .The half time versus initial monomer concentration plots for the apoA-I 1-83 variants, presented as double logarithmic graphs, reveal a linear decrease in half time at low monomer concentrations, while the slope becomes flat at high monomer concentrations (Fig. 5C).This monomer concentration-independent behavior at the half time suggests the saturation of the elongation step at high monomer concentrations 33,34 .The apparent rate constants for fibril elongation increase at low monomer concentrations but remain unchanged at high monomer concentrations for all 1-83 variants (Fig. 5D-G).In contrast, the lag time tends to decrease with increasing monomer concentration, except for the Δ60-71/VT variant, where the lag time remains constant across all examined concentrations (Fig. 5D-G).We also applied the online Amylofit program (https:// www.amylo fit.ch.cam.ac.uk) 32 to the kinetic data shown in Fig. 5A, B with a model involving saturating elongation and secondary nucleation.From the double logarithmic plot of half time and initial monomer concentration, which gives the scaling exponent, γ, as the slop of the plot, we calculated the reaction order of secondary nucleation, n 2 , and was kept constant in the fitting process, while other kinetic parameters were allowed to vary.Best fitted results were shown in Fig. S4 in the Supplementary information.From the combined kinetic parameters for primary nucleation and elongation (k n k + ) or secondary nucleation and elongation (k 2 k + ), we obtained the effective noncatalytic fibril proliferation rates through primary and secondary processes, λ and κ, respectively.Given the relative magnitude of λ and κ determines the dominant process in the overall aggregation 35 , the finding of significant lower values κ compared λ indicates that the primary nucleation and elongation process is dominant over the secondary processes in apoA-I fibril formation.
We further investigated the impact of preformed seeds on the kinetics of fibril formation for apoA-I 1-83 variants.It is widely recognized that the presence of preformed seeds strongly accelerates fibril formation by bypassing the conversion of soluble proteins into amyloid nuclei [35][36][37] .As depicted in Fig. 6A, B, the intensity of ThT fluorescence increased rapidly for the 1-83 and Δ60-71/VT variants with increasing concentrations of seed fibrils.Moreover, an increase in seed concentration gradually reduced the lag time without significantly affecting the apparent rate constant of fibril elongation, ultimately leading to the near elimination of the lag time beyond 5 μg/ml of seeds (Fig. 6C, D).This observation suggests that primary nucleation is the predominant process during the lag phase of fibril formation in the apoA-I 1-83 variants 35 .

Thermodynamic analysis of fibril formation of apoA-I 1-83 variants
We conducted a thermodynamic analysis of the fibril-forming characteristics of apoA-I 1-83 variants by monitoring the kinetics of fibril formation at various temperatures 25,38,39 .We note that there are no secondary structural changes across the temperature range at which the thermodynamic analysis was performed 25 .Figure 7A shows the time course of ThT fluorescence intensities for apoA-I 1-83, with the curves fitted using the Finke-Watzky equation at different temperatures.The obtained rate constants for nucleation (k 1 ) and fibril elongation (k 2 ) increased as the temperature rose (Fig. 7C).Based on the linear plots derived from the Eyring equation for each rate constants of k 1 and k 2 (Fig. 7E), we determined the activation enthalpy (ΔH * ) and entropy (ΔS * ) for the nucleation and fibril elongation steps in fibril formation by apoA-I 1-83.As shown in Table 2, a comparison of the ΔH * and ΔS * values together with the activation Gibbs free energy (ΔG * ) values demonstrated that both the nucleation and fibril elongation steps in the fibril formation of apoA-I 1-83 were enthalpically and entropically unfavorable.
Similarly, amyloidogenic Δ60-71/VT, Δ70-72, and F71Y variants of apoA-I 1-83 exhibited temperaturedependent increases in ThT fluorescence intensity (Fig. 7B and Supplementary Fig. S5A, B).The Eyring plots of the rate constants k 1 and k 2 (Fig. 7F and Supplementary Fig. S5C, D) provided the thermodynamic parameters for each variant (Table 2).In sharp contrast to the wild-type apoA-I 1-83, the Δ60-71/VT variant showed a significant reduction in the unfavorable activation entropy and a concomitant increase in the unfavorable enthalpy for nucleation in fibril formation.For fibril elongation, the unfavorable activation enthalpy and entropy values for the Δ60-71/VT variant were similar to those of the wild-type apoA-I 1-83.These results indicate that the Δ60-71/VT mutation promoted fibril formation in apoA-I 1-83 through reducing entropic barrier for the nucleation process.As for the Δ70-72 and F71Y variants, similarly unfavorable activation enthalpy and entropy values were observed for both nucleation and fibril elongation, compared to those of the wild-type apoA-I 1-83.We note that the constancy of the ΔG * values for the nucleation and fibril elongation of all the variants indicates the occurrence of the enthalpy-entropy compensation effects [40][41][42] .

Discussion
Many human exchangeable apolipoproteins, including apoA-I, are known as amyloid precursor proteins involved in hereditary or acquired forms of disease 44,45 .These apolipoproteins have partially folded dynamic conformations in the lipid-free state.In apoA-I, the N-terminal helix bundle domain contains two major aggregation-prone segments of residues 14-22 and 53-58, as well as a minor segment of residues 69-72 18,25 .It has been proposed that amyloidogenic mutations that structurally destabilize apoA-I increase the exposure of these aggregationprone segments, leading to protein aggregation and fibril formation 17,46 .However, the present results (Fig. 1) indicate that structural destabilization and exposure of hydrophobic surfaces in the N-terminal helix bundle alone are not sufficient to trigger fibril formation by full-length apoA-I at neutral pH, consistent with previous studies 12,47 .Other factors such as oxidative modification of methionine residues [48][49][50] , proteolysis 51 , or interactions with amyloid-associated proteins 52 or heparan sulfate 53,54 may be required to initiate the conversion of full-length apoA-I into the amyloid fibrillar form at neutral pH.
In contrast to the full-length form, the N-terminal 1-83 fragment of apoA-I, which predominantly exists as a random coil structure in solution shows a strong propensity to form amyloid fibrils at neutral pH 12 .Certain amyloidogenic mutations can enhance the aggregation and fibril formation of apoA-I 1-83 fragments in the aqueous phase 12 , on lipid membranes 43,55 , or in the presence of heparin 56 .Our recent findings demonstrated that the two major aggregation-prone segments, residues 14-22 and 53-58, play critical roles in formation of the G26R variant of apoA-I 1-83 fragment.Residues 14-22 are necessary β-transition and fibril formation, whereas residues 53-58 entropically nucleation 25 .Consistently, these two amyloidogenic segments found to be in close proximity, forming amyloid core structures in apoA-I 1-83/G26R fibrils 25 .
An analysis of the of fibril-forming kinetics on the monomer concentration of apoA-I 1-83 variants reveals that saturation of the elongation step occurs at high monomer concentrations (Fig. 5).It has been suggested that, at sufficiently high protein concentrations, rate-determining step for elongation involves a transition from the diffusive attachment of a monomer to fibril end to the structural rearrangement of the incorporated into the fibril.This transition is independent the monomer concentration 33,37 .The parallel shifts observed in the double logarithmic plots for all apoA-I variants (Fig. 5C) indicate that such saturation of the elongation step occurs, despite the acceleration of the nucleation step 33,57 .Furthermore, experiments on seeded aggregation kinetics demonstrate that the length of the lag phase decreases with increasing seed concentration, without substantially affecting the fibril elongation rate (Fig. 6).This finding suggests that primary nucleation is the dominant process during lag phase of fibril in 1-83 variants.It is possible that the relatively less hydrophobic characteristics of the apoA-I 1-83 fragment, along with the presence of the negatively charged amino acid-rich C-terminal region 43 , favor primary nucleation over surface-catalyzed secondary nucleation processes 34,58 .
A significant finding of this study was that the Δ60-71/VT mutation, among the amyloidogenic mutations occurring near the aggregation-prone segment of residues 67-72, significantly enhanced nucleation and fibril elongation during the formation of fibrils by the apoA-I 1-83 fragment (Fig. 2 and Supplementary Fig. S1).(VTSTFSKVTW).This newly formed aggregation-prone segment is comparable to the largest aggregation-prone segment of residues 14-22, which is necessary for formation in the apoA-I 1-83 fragment 25 .Thermodynamic analyses of fibril formation kinetics 7 and Supplementary Fig. S5) gave further insights into the aggregation mechanism of apoA-I 1-83 variants.As in this study (Table 2), it was reported that the activation enthalpies of nucleation and fibril elongation are unfavorable for many amyloidogenic proteins, likely because the net unfavorable and breakage of many weak interactions are necessary to reach the transition state 38,59,60 .Regarding the activation entropy of nucleation, in contrast, we found in the present study that the Δ60-71/VT variant exhibits an almost eliminated activation entropy for nucleation during fibril formation, unlike the large unfavorable activation entropy observed in other variants (Table 2).In the previous studies, the favorable activation entropy of was in G26R of the apoA-I 1-83 fragment 25 and β-amyloid 42 38 , likely arising from the desolvation of amyloidogenic regions in the protein molecule at the transition state 25,38,60 .In this regard, desolvation of the large aggregation-prone segment around residues 53-62 in the Δ60-71/VT variant may contribute to an elimination of entropic barrier for nucleation, providing a template for the intermolecular β-sheet formation 10 .
Regarding fibril elongation, the Δ60-71/VT variant exhibits similar unfavorable activation entropy for fibril elongation to those of other apoA-I 1-83 variants (Table 2).Since the rate of fibril formation is associated with the mechanical stability of the fibril state, faster protein aggregation likely results in a fibrillar state with higher mechanical stability 61 .Therefore, it is plausible that the increased stability of the fibrils formed by the Δ60-71/VT variant, compared to the other 1-83 variants (Fig. 4B), is linked to its faster fibril elongation.It should be noted that the enhanced stability of the Δ60-71/VT fibrils against urea-induced disaggregation may be attributed to the distinct amyloid core structures composed of highly amyloidogenic segments in the apoA-I 1-83 fragment 25 .
In summary, we have demonstrated, for the first time, that the amyloidogenic Δ60-71/VT mutation significantly enhances the formation of fibrils in the N-terminal 1-83 fragment of apoA-I.Specifically, the Δ60-71/ VT mutation generates a large aggregation-prone segment spanning residues 53-62, which plays a crucial role in promoting the nucleation process during fibril formation.This aggregation-prone segment promotes the nucleation through reducing entropic barrier, likely serving as a template for nucleation of intermolecular aggregation.These findings emphasize the pivotal role of amyloidogenic mutations in apoA-I in the intermolecular aggregation and nucleation of its N-terminal fragment.

Preparation of recombinant apoA-I proteins and peptides
The thioredoxin (Trx)-fused wild-type N-terminal 1-83 fragments of apoA-I and amyloidogenic Δ60-71/VT, Δ70-72, F71Y variants expressed in E. coli were purified as previously described 12 .Trx was cleaved by thrombin, which produced apoA-I fragments with two extra N-terminus amino acids, Gly-Ser.The apoA-I preparations were at least 95% pure, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The apoA-I 50-75 (WDSVTSTFSKLREQLGPVTQEFWDNL), 50-75 Δ60-71 (WDSVTSTFSKWDNL), and 50-75 Δ60-71/ VT (WDSVTSTFSKVTWDNL) peptides were synthesized by a solid-phase method using Fmoc chemistry as described 24 .The N-and C-termini were capped with acetyl and amide groups, respectively.All apoA-I fragments were solubilized in a 6 M GdnHCl solution, which was dialyzed into the appropriate buffer before use.

Preparation of seed fibrils
Seed fibrils were produced by incubating μg/ml of apoA-I solution at 37 ºC with shaking, followed by centrifugation at 20,000g for 40 min.Fibril pellets were collected and resuspended in 10 mM Tris buffer (150 mM NaCl, 0.02% NaN 3 , pH 7.4) using a Branson bath-type sonicator for 1 min.The resultant seed fibril solution was stored at 4 °C, and continuously sonicated for 1 min before use.

ATR-FTIR spectrometry
A Jasco FTIR spectrometer FT/IR-4700 equipped with an ATR PKM-Ge-L reflectance accessory was used to record the ATR-FTIR spectra.An aliquot of the apoA-I peptide samples (1.4 mg/ml) in Tris buffer (pH 7.4) was spread on a germanium waveguide and dried under flowing nitrogen gas.ATR-FTIR spectra in the wavenumber range of 1000-3500 cm -1 were obtained at a resolution of 4 cm -1 with 256 accumulations under continuous nitrogen purging.

Fluorescence measurements
Fluorescence measurements were performed on an F-7000 fluorescence spectrophotometer (Hitachi High-Technologies, Tokyo, Japan) and an infinite 200 PRO plate reader (TECAN) at 25 °C.To monitor the exposure of hydrophobic sites on the apoA-I variants, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectra were collected from 400 to 600 nm at an excitation wavelength of 395 nm in the presence of 50 μg/ml protein and an excess of ANS (250 μM).The kinetics of amyloid fibril formation was monitored by measuring the fluorescence intensities of ThT 63 .Solutions of the apoA-I 1-83 variants or peptides (200 μg/mL) in Tris buffer (pH 7.4) were incubated and shaken at 37 ºC on a microplate shaker in the presence of 10 μM ThT.Time-dependent increases in ThT fluorescence intensity were fitted to the following sigmoidal equation 25,64 : where F is the fluorescence intensity, F 0 and F max are the initial and final baselines during the lag and plateau phases, respectively.k app the apparent rate constant for fibril elongation and t m is the time to 50% of maximal fluorescence.The lag time is given as t m − 2/k.
We the ThT fluorescence data using the Finke-Watzky equation for a two-step model of nucleation followed by autocatalytic growth 30,65 : where [A] 0 is the initial protein concentration, and k 1 and k 2 are the rate constants for nucleation and fibril elongation, respectively.The thermodynamic parameters for nucleation and fibril elongation were determined using the Eyring equation: where k B is the Boltzmann constant and h is the Planck constant.The slope and y-intercept of the linear plot according to Eq. ( 3) give the activation enthalpy (ΔH * ) and entropy (ΔS * ), respectively.The activation Gibbs free energy (ΔG * ) was obtained from ΔH * and ΔS * according to ∆G * = ∆H * -T∆S * .
To compare the stability of the fibrils against denaturant-induced disaggregation, fibrils formed by apoA-I 1-83 variants (50 μg/ml in Tris buffer, pH 7.4) were incubated overnight at 4 °C with various concentrations of urea in the presence of ThT (10 μM), during which ThT fluorescence intensities were monitored as described above.

AFM
AFM was performed as previously described 25 .Briefly, samples were deposited on freshly cleaved mica, and AFM images were obtained under ambient conditions at room temperature using a NanoScope ® IIIa Tapping mode AFM (Veeco, Plainview, NY) and a micro cantilever OMCL-AC160TS-R3 (Olympus, Tokyo, Japan).

TEM and TIRFM
TEM and TIRFM were performed as previously described 39,66 .Briefly, for TEM, the samples were negatively stained with a phosphomolybdic acid solution, and TEM measurements were performed using a JEOL JEM-1200EX transmission microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.ThT fluorescence images were obtained using an inverted microscope (IX70; Olympus, Tokyo, Japan).An argon laser was used to excite the ThT.The signals were cleaned using using a band-pass filter and visualized using an SIT camera equipped with an image intensifier.

Cytotoxicity assay
The cytotoxicity of fibrils formed by apoA-I fragments against HEK293 cells was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described 12 .Briefly, HEK293 cells were plated and grown on poly-l-lysine-coated 24-well plates in DMEM containing 2% Fetal Bovine Serum for 24 h, after which they were cultured in the presence of fibrils formed by apoA-I 1-83 variants in 10 mM phosphate-buffered saline (pH 7.4) for 24 h.Cell viability was quantitatively determined by optically measuring the reduction of MTT to formazan by living cells.

Statistical analysis
We analyzed the data by means of one-way ANOVA with the Dunnett's test or the Tukey's multiple comparisons test.Results were considered significant at P < 0.05. (1)

Figure 1 .
Figure 1.Effects of amyloidogenic mutations on the structural stability and fibril-forming propensity of full-length apoA-I.(A) CD spectra of apoA-I wild-type (WT), Δ60-71/VT, Δ70-72, and F71Y.(B) Thermal unfolding of apoA-I variants monitored by the ellipticity at 222 nm.(C) ANS fluorescence spectra in the presence of apoA-I variants.ANS fluorescence spectrum of free ANS in buffer was shown for comparison.a. u., arbitrary units.(D) ThT fluorescence intensity for apoA-I WT (open circle), Δ60-71/VT (open triangle), Δ70-72 (filled reverse triangle), and F71Y (open square) were plotted as a function of time.The data for apoA-I 1-83 fragment (open diamond) was shown for comparison.Protein and ThT concentrations were 200 μg/ml and 10 μM, respectively.a. u., arbitrary units.

Table 1 .
α-Helix content, thermal denaturation parameters, and ANS binding for full-length apoA-I variants.aMean± SD from at least three independent experiments.bThereproducibility in T m is ± 1.3 ºC.c Calculated as described under "Materials and methods".d Estimated error is within ± 4 kJ/mol.e Values are ratios of wild-type apoA-I.Estimated error is within ± 0.1.